A variety of methods for forming peptide bonds have been described in the literature and used in laboratories and by peptide suppliers. Included among these are the use of anhydrides of amino acids (particularly mixed carboxylic-carbonic anhydrides as well as symmetrical anhydrides of acylamino acids), the use of activated esters of amino acids (such as p-nitrophenyl esters, 3,4,5-trichlorophenyl esters, and pentafluorophenyl esters), and the use of coupling reagents (such as dicyclohexylcarbodiimide, diisopropylcarbodiimide, and carbodiimides bearing tertiary amine or quaternary ammonium groups).
Each of these methods has its own complications that limit either the yield, the reaction rate, or the economy or the efficiency of its use. A method that would appeared to avoid many of these complications is the use of N-carboxyanhydrides of amino acids as reagents. The appeal of N-carboxyanhydrides is the fact that they are easily prepared in one step with a negligible waste stream, their reactivity is high enough that they couple rapidly at room temperature with nitrogen nucleophiles, and the coupling by-product is carbon dioxide. The high reactivity however causes an inherent instability, a propensity for polymerization, and a tendency for loss of enantiomeric integrity.
Researchers have attempted to overcome these deficiencies by using an N-carboxyanhydride in which the nitrogen is additionally substituted with tosyl (p-toluenesulfonyl) (Zaoral, M., et al, Collect. Czech. Chem. Comun. 1961, 26, 2316) and nitrophenysulfonyl groups (Kricheldorf, H. R., Angew. Chem. 1973, 85, 86; Halstrom, J., et al., Acta Chem. Scand., Ser. B 1979, B33, 685), with little improvement. The most promising results were achieved recently with an alkoxycarbonyl group added to the nitrogen, forming a urethane-protected-N-carboxyanhydride (Fuller, W. D., et al, J. Am. Chem. Soc. 1990, 112, 7414). While this modification reduced the instability and tendency to polymerize, there still remains a significant loss of enantiomeric purity during peptide synthesis (Romoff, T. T., et al., Peptide Res., 1997, 49, 281) and some instability to base.
A report of the use of N-trityl N-carboxyanhydrides in peptide synthesis appeared in 1962 (Block, H., et al., Proceedings of the 5.sup.th European Peptide Symp. 1962, pp. 83-87). The N-trityl N-carboxyanhydrides used in the syntheses were those of glycine and alanine. Neither of these are suitable amino acids for studying the concerns of N-carboxyanhydrides, since glycine lacks a side chain and hence an asymmetric center at its a-carbon, and alanine has a methyl group as its side chain which renders it highly stable relative to amino acids with side chains containing electron-withdrawing groups. Also, the report lacks proper confirmatory analyses of the products.